SRM/MRM assay for the 6-O-methylguanine-DNA methyltransferase (MGMT) protein

ABSTRACT

The current disclosure provides methods for detecting and quantitating the 6-O-methylguanine-DNA methyltransferase protein (MGMT) directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM) mass spectrometry. Such biological samples are chemically preserved and fixed with formaldehyde containing agents/fixatives and may include formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and/or paraffin embedded. A protein sample is prepared from the biological sample and the MGMT protein is quantitated in the sample using SRM/MRM mass spectrometry by quantitating one or more fragment peptides.

This application claims priority to provisional application Ser. No.62/161,651, filed May 14, 2015, the contents of which are herebyincorporated by reference in their entirety. This application alsocontains a sequence listing submitted electronically via EFS-web, whichserves as both the paper copy and the computer readable form (CRF) andconsists of a file entitled “001152_8044_US01_SEQ_LISTING”, which wascreated on May 13, 2016, which is 849 bytes in size, and which is alsoincorporated by reference in its entirety.

INTRODUCTION

Cancer is treated with a collection of therapeutic agents that killgrowing and dividing cells and that function in a variety of ways. Acommon collection of chemotherapeutic agents has been used for decades,either individually or in combinations, and this common collection ofagents has become the traditional and routine cancer treatment inclinical oncology practice. These traditional chemotherapeutics agentsact by killing all cells that divide rapidly, rapid cell division beingone of the main properties of most cancer cells. However, these agentsalso kill growing normal cells and, accordingly, these agents are notconsidered to be “targeted” approaches to killing cancer cells. Inrecent years a large group of cancer therapeutic agents has beendeveloped that selectively target only cancer cells where thetherapeutic agent specifically attacks a protein that either is onlyexpressed by the cancer cells and not by normal cells, or that isexpressed in much higher amounts in cancer cells compared to normalcells. This approach is considered to be a “targeted” approach to cancertherapy. Most recently, another approach to killing cancer cells in a“targeted” fashion has been to specifically modulate the immune systemto enhance that ability of the cancer patient's immune system to killcancer cells.

Therapeutic agents that target the 6-O-methylguanine-DNAmethyltransferase protein, which is also referred to as MGMT, have shownpromise in early clinical trials. However, only those patients whosecancer cells express high amounts of the MGMT protein would be mostlikely to benefit from treatment with these MGMT-targeted therapeuticagents. The methods described below provide a quantitativeproteomics-based assay that delivers a relevant measure of activation ofthe MGMT signal pathway as MGMT is not normally expressed in normaltissue and/or normal epithelial cells. In particular, the methodsprovide a mass spectrometry assay that quantifies MGMT in formalin fixedtissues from cancer patients and that enables improved treatmentdecisions for cancer therapy.

Specific peptides derived from subsequences of the 6-O-methylguanine-DNAmethyltransferase protein, also referred to as MGMT,methylated-DNA-protein-cysteine methyltransferase, and referred toherein as MGMT, are provided. The peptide sequence andfragmentation/transition ions for each peptide are particularly usefulin a mass spectrometry-based Selected Reaction Monitoring (SRM), whichcan also be referred to as a Multiple Reaction Monitoring (MRM) assay,and will be referred to as SRM/MRM. The use of peptides for SRM/MRMquantitative analysis of the MGMT protein is described.

This SRM/MRM assay can be used to measure relative or absolutequantitative levels of one or more of the specific peptides from theMGMT protein and therefore provide mass spectrometric methods ofmeasuring by mass spectrometry the amount of the MGMT protein in a givenprotein preparation obtained from a biological sample.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by references in their entirety. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue reagents and protocol available from Expression Pathology Inc.(Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatients tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue are by farthe most common method of preserving cancer tissue samples worldwide andis the accepted convention for standard pathology practice. Aqueoussolutions of formaldehyde are referred to as formalin. “100%” formalinconsists of a saturated solution of formaldehyde (about 40% by volume or37% by mass) in water, with a small amount of stabilizer, usuallymethanol to limit oxidation and degree of polymerization. The mostcommon way in which tissue is preserved is to soak whole tissue forextended periods of time (8 hours to 48 hours) in aqueous formaldehyde,commonly termed 10% neutral buffered formalin, followed by embedding thefixed whole tissue in paraffin wax for long term storage at roomtemperature. Thus molecular analytical methods to analyze formalin fixedcancer tissue will be the most accepted and heavily utilized methods foranalysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate andprecise quantitative levels of the MGMT protein within the specifictissue samples (e.g., cancer tissue sample) of the patient or subjectfrom whom the tissue (biological sample) was collected and preserved.This not only provides diagnostic and prognostic information about thecancer, but also permits a physician or other medical professional tomore accurately determine appropriate therapy for the patient. Such anassay that provides diagnostically, prognostically, and therapeuticallyimportant information about levels of protein expression in a diseasedtissue or other patient sample is termed a companion diagnostic assay.For example, such an assay can be designed to diagnose the stage ordegree of a cancer and determine a therapeutic agent to which a patientis most likely to respond.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the MGMT protein and also can measureabsolute or relative levels of specific modified peptides from the MGMTprotein. Examples of modifications include phosphorylated amino acidresidues and glycosylated amino acid residues that may be present on thepeptides.

Relative quantitative levels of the MGMT protein are determined by theSRM/MRM methodology for example by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual MGMT peptide in different samples. Alternatively, it ispossible to compare multiple SRM/MRM signature peak areas for multipleMGMT signature peptides, where each peptide has its own specific SRM/MRMsignature peak, to determine the relative MGMT protein content in onebiological sample with the MGMT protein content in one or moreadditional or different biological samples. In this way, the amount of aparticular peptide, or peptides, from the MGMT protein, and thereforethe amount of the MGMT protein, is determined relative to the same MGMTpeptide, or peptides, across two or more biological samples under thesame experimental conditions. In addition, relative quantitation can bedetermined for a given peptide, or peptides, from the MGMT proteinwithin a single sample by comparing the signature peak area for thatpeptide by SRM/MRM methodology to the signature peak area for anotherand different peptide, or peptides, from a different protein, orproteins, within the same protein preparation from the biologicalsample. In this way, the amount of a particular peptide from the MGMTprotein, and therefore the amount of the MGMT protein, is determinedrelative one to another within the same sample. These approaches permitquantitation of an individual peptide, or peptides, from the MGMTprotein to the amount of another peptide, or peptides, between samplesand within samples wherein the amounts as determined by signature peakarea are relative one to another, regardless of the absolute weight tovolume or weight to weight amounts of the MGMT peptide in the proteinpreparation from the biological sample. Relative quantitative data aboutindividual signature peak areas between different samples are normalizedto the amount of protein analyzed per sample. Relative quantitation canbe performed across many peptides from multiple proteins and the MGMTprotein simultaneously in a single sample and/or across many samples togain insight into relative protein amounts of one peptide/protein withrespect to other peptides/proteins.

Absolute quantitative levels of the MGMT protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the MGMT protein in one biological sampleis compared to the SRM/MRM signature peak area of a spiked internalstandard. In one embodiment, the internal standard is a syntheticversion of the same exact MGMT peptide that contains one or more aminoacid residues labeled with one or more heavy isotopes. Such an isotopelabeled internal standard is synthesized so that when analyzed by massspectrometry it generates a predictable and consistent SRM/MRM signaturepeak that is different and distinct from the native MGMT peptidesignature peak and which can be used as a comparator peak. Thus when theinternal standard is spiked into a protein preparation from a biologicalsample in known amounts and analyzed by mass spectrometry, the SRM/MRMsignature peak area of the native peptide is compared to the SRM/MRMsignature peak area of the internal standard peptide, and this numericalcomparison indicates either the absolute molarity and/or absolute weightof the native peptide present in the original protein preparation fromthe biological sample. Absolute quantitative data for fragment peptidesare displayed according to the amount of protein analyzed per sample.Absolute quantitation can be performed across many peptides, and thusproteins, simultaneously in a single sample and/or across many samplesto gain insight into absolute protein amounts in individual biologicalsamples and in entire cohorts of individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, and/or the patient prognosis, for example, directly inpatient-derived tissue, such as formalin fixed tissue, and to aid indetermining which therapeutic agent would be most advantageous for usein treating that patient. Cancer tissue that is removed from a patienteither through surgery, such as for therapeutic removal of partial orentire tumors, or through biopsy procedures conducted to determine thepresence or absence of suspected disease, is analyzed to determinewhether or not a specific protein, or proteins, and which forms ofproteins, are present in that patient tissue. Moreover, the expressionlevel of a protein, or multiple proteins, can be determined and comparedto a “normal” or reference level found in healthy tissue. Normal orreference levels of proteins found in healthy tissue may be derivedfrom, for example, the relevant tissues of one or more individuals thatdo not have cancer. Alternatively, normal or reference levels may beobtained for individuals with cancer by analysis of relevant tissues notaffected by the cancer. Assays of protein levels (e.g., MGMT levels) canalso be used to diagnose, and provide prognostic information about, thestage of cancer in a patient or subject diagnosed with cancer byemploying the MGMT levels. The level of an individual MGMT peptide isdefined as the molar amount of the peptide determined by the SRM/MRMassay per total amount of protein lysate analyzed. Information regardingMGMT can thus be used to aid in determining the stage or grade of acancer by correlating the level of the MGMT protein (or fragmentpeptides of the MGMT protein) with levels observed in normal tissues.Once the quantitative amount of the MGMT protein has been determined inthe cancer cells, that information can be matched to a list oftherapeutic agents (chemical and biological) developed to specificallytreat cancer tissue that is characterized by, for example, abnormalexpression of the protein or protein(s) (e.g., MGMT) that were assayed.Matching information from an MGMT protein assay to a list of therapeuticagents that specifically targets, for example, the MGMT protein orcells/tissue expressing the protein, defines what has been termed apersonalized medicine approach to treating disease. The assay methodsdescribed herein form the foundation of a personalized medicine approachby using analysis of proteins from the patient's own tissue as a sourcefor diagnostic and treatment decisions.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from the MGMT protein,prepared for example by digesting with a protease of known specificity(e.g. trypsin), can be used as a surrogate reporter to determine theabundance of MGMT protein in a sample using a mass spectrometry-basedSRM/MRM assay. Similarly, any predicted peptide sequence containing anamino acid residue at a site that is known to be potentially modified inthe MGMT protein also might potentially be used to assay the extent ofmodification of the MGMT protein in a sample.

MGMT fragment peptides may be generated by a variety of methodsincluding by the use of the Liquid Tissue protocol provided in U.S. Pat.No. 7,473,532. The Liquid Tissue protocol and reagents are capable ofproducing peptide samples suitable for mass spectroscopic analysis fromformalin fixed paraffin embedded tissue by proteolytic digestion of theproteins in the tissue/biological sample. In the Liquid Tissue protocolthe tissue/biological is heated in a buffer for an extended period oftime (e.g., from about 80° C. to about 100° C. for a period of time fromabout 10 minutes to about 4 hours) to reverse or release proteincross-linking. The buffer employed is a neutral buffer, (e.g., aTris-based buffer, or a buffer containing a detergent). Following heattreatment the tissue/biological sample is treated with one or moreproteases, including but not limited to trypsin, chymotrypsin, pepsin,and endoproteinase Lys-C for a time sufficient to disrupt the tissue andcellular structure of said biological sample and to liquefy the sample(e.g. a period of time from 30 minutes to 24 hours at a temperature from37° to 65° C.). The result of the heating and proteolysis is a liquid,soluble, dilutable biomolecule lysate.

Surprisingly, it was found that many potential peptide sequences fromthe MGMT protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. This was especially true for peptides derived from formalinfixed tissue. As it was not possible to predict the most suitablepeptides for MRM/SRM assay, it was necessary to experimentally identifymodified and unmodified peptides in actual Liquid Tissue lysates todevelop a reliable and accurate SRM/MRM assay for the MGMT protein.While not wishing to be bound by any theory, it is believed that somepeptides might, for example, be difficult to detect by mass spectrometrybecause they do not ionize well or produce fragments distinct from otherproteins. Peptides may also fail to resolve well in separation (e.g.,liquid chromatography), or may adhere to glass or plastic ware.

MGMT peptides found in various embodiments of this disclosure (e.g.,Tables 1 and 2) were derived from the MGMT protein by protease digestionof all the proteins within a complex Liquid Tissue lysate prepared fromcells procured from formalin fixed cancer tissue. Unless notedotherwise, in each instance the protease was trypsin. The Liquid Tissuelysate was then analyzed by mass spectrometry to determine thosepeptides derived from the MGMT protein that are detected and analyzed bymass spectrometry. Identification of a specific preferred subset ofpeptides for mass-spectrometric analysis is based on; 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue lysates, and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissuereagents and protocol that entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, as forexample including but not limited to the protease trypsin. Each proteinlysate is turned into a collection of peptides by digestion of intactpolypeptides with the protease. Each Liquid Tissue lysate was analyzed(e.g., by ion trap mass spectrometry) to perform multiple globalproteomic surveys of the peptides where the data was presented asidentification of as many peptides as could be identified by massspectrometry from all cellular proteins present in each protein lysate.An ion trap mass spectrometer or another form of a mass spectrometerthat is capable of performing global profiling for identification of asmany peptides as possible from a single complex protein/peptide lysateis typically employed. Ion trap mass spectrometers however may be thebest type of mass spectrometer for conducting global profiling ofpeptides. Although an SRM/MRM assay can be developed and performed onany type of mass spectrometer, including a MALDI, ion trap, or triplequadrupole, advantageously the instrument platform for an SRM/MRM assayis a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single MSanalysis of a single lysate under the conditions employed, then thatlist of peptides was collated and used to determine the proteins thatwere detected in that lysate. That process was repeated for multipleLiquid Tissue lysates, and the very large list of peptides was collatedinto a single dataset. That type of dataset can be considered torepresent the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the MGMT protein.

In one embodiment, the MGMT tryptic peptides identified as useful in thedetermination of absolute or relative amounts of the MGMT proteininclude one or more of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, eachof which are listed in Table 1. Each of these peptides was detected bymass spectrometry in Liquid Tissue lysates prepared from formalin fixed,paraffin embedded tissue. Thus, each peptide is a candidate for use indeveloping a quantitative SRM/MRM assay for the MGMT protein in humanbiological samples, including directly in formalin fixed patient tissue.

TABLE 1 Pre- Mono cursor Pre- Tran- Peptide Isotopic Charge cursorsition Ion sequence Mass State m/z m/z Type FGEVISYQ 1904.9995   2953.507  486.267 y5 QLAALAGN   2 953.507  599.351 y6 PK   2 953.507 670.388 y7   2 953.507  741.425 y8   2 953.507  854.509 y9   2 953.507 982.567 y10   2 953.507 1110.626 y11   2 953.507 1273.689 y12   2953.507 1360.721 y13   2 953.507 1473.805 y14 GAGATSGS 1084.5261   2543.27   303.177 y3 PPAGR   2 543.27   400.23  y4   2 543.27   497.283y5   2 543.27   584.315 y6   2 543.27   641.336 y7   2 543.27   728.368y8   2 543.27   829.416 y9   2 543.27   900.453 y10   2 543.27   957.474y11   2 543.27  1028.511 y12 EWLLAHEG 1246.6206   2 624.318  369.199 y3HR   2 624.318  498.241 y4   2 624.318  635.3   y5   2 624.318  706.337y6 8 2 624.318  819.421 y7   2 624.318  932.506 y8   2 624.318 1118.585y9The MGMT tryptic peptides listed in Table 1 were detected from multipleLiquid Tissue lysates of multiple different formalin fixed tissues ofdifferent human organs including prostate, colon, and breast. Each ofthose peptides is considered useful for quantitative SRM/MRM assay ofthe MGMT protein in formalin fixed tissue. Further data analysis ofthese experiments indicated no preference is observed for any of thesepeptides from any specific organ site. Thus, each of these peptides isbelieved to be suitable for conducting SRM/MRM assays of the MGMTprotein on a Liquid Tissue lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

An important consideration when conducting an SRM/MRM assay is the typeof instrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for an SRM/MRM assay ispresently most likely considered to be a triple quadrupole instrumentplatform. That type of a mass spectrometer is presently the mostsuitable instrument for putative analysis of a single isolated targetpeptide within a very complex protein lysate that may consist ofhundreds of thousands to millions of individual peptides from all theproteins contained within a cell.

In order to most efficiently implement an SRM/MRM assay for each peptidederived from the MGMT protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer) to perform thecorrect and focused analysis of specific targeted peptide(s), such thatthe assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific MGMT peptides, may include one or more of the mono isotopicmass of the peptide, its precursor charge state, the precursor m/zvalue, the m/z transition ions, and the ion type of each transition ion.Additional peptide information that may be used to develop an SRM/MRMassay for the MGMT protein is shown for these MGMT peptides in Table 1.

The method described below was used to: 1) identify candidate peptidesfrom the MGMT protein that can be used for a mass spectrometry-basedSRM/MRM assay for the MGMT protein, 2) develop an individual SRM/MRMassay, or assays, for target peptides from the MGMT protein in order tocorrelate and 3) apply quantitative assays to cancer diagnosis and/orchoice of optimal therapy.

Assay Method

1. Identification of SRM/MRM candidate fragment peptides for the MGMTprotein

-   -   a. Prepare a Liquid Tissue protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the MGMT protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the MGMT protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length MGMT protein potentially can be        measured, but preferred peptides used for development of the        SRM/MRM assay are those that are identified by mass spectrometry        directly in a complex Liquid Tissue protein lysate prepared from        a formalin fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the        MGMT protein        2. Mass Spectrometry Assay for Fragment Peptides from the MGMT        Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue        lysate is applied to peptides from the MGMT protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the MGMT protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the protein in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                MGMT protein by comparing the SRM/MRM signature peak                area from a given MGMT peptide detected in a Liquid                Tissue lysate from one formalin fixed biological sample                to the same SRM/MRM signature peak area of the same MGMT                fragment peptide in at least a second, third, fourth or                more Liquid Tissue lysates from least a second, third,                fourth or more formalin fixed biological samples            -   2. Determining increased or decreased presence of the                MGMT protein by comparing the SRM/MRM signature peak                area from a given MGMT peptide detected in a Liquid                Tissue lysate from one formalin fixed biological sample                to SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                MGMT protein by comparing the SRM/MRM signature peak                area for a given MGMT peptide to the SRM/MRM signature                peak areas from other fragment peptides derived from                different proteins within the same Liquid Tissue lysate                from the formalin fixed biological sample in order to                normalize changing levels of MGMT protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the MGMT protein, where the modifications include but                are not limited to phosphorylation and/or glycosylation,                and where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the MGMT protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the MGMT protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.                3. Apply Fragment Peptide Quantitation to Cancer                Diagnosis and Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the MGMT protein and demonstrate that the        previously-determined association, as well understood in the        field of cancer, of MGMT protein expression to the        stage/grade/status of cancer in patient tumor tissue is        confirmed    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the

MGMT protein and demonstrate correlation with clinical outcomes fromdifferent treatment strategies, wherein this correlation has alreadybeen demonstrated in the field or can be demonstrated in the futurethrough correlation studies across cohorts of patients and tissue fromthose patients. Once either previously established correlations orcorrelations derived in the future are confirmed by this assay then theassay method can be used to determine optimal treatment strategy

Specific and unique characteristics about specific MGMT peptides weredeveloped by analysis of all MGMT peptides on both an ion trap andtriple quadrupole mass spectrometers. That information includes themonoisotopic mass of the peptide, its precursor charge state, theprecursor m/z value, the transition m/z values of the precursor, and theion types of each of the identified transitions. That information mustbe determined experimentally for each and every candidate SRM/MRMpeptide directly in Liquid Tissue lysates from formalin fixedsamples/tissue; because, interestingly, not all peptides from the MGMTprotein can be detected in such lysates using SRM/MRM as describedherein, indicating that MGMT peptides not detected cannot be consideredcandidate peptides for developing an SRM/MRM assay for use inquantitating peptides/proteins directly in Liquid Tissue lysates fromformalin fixed samples/tissue.

A particular SRM/MRM assay for a specific MGMT peptide is performed on atriple quadrupole mass spectrometer. An experimental sample analyzed bya particular MGMT SRM/MRM assay is for example a Liquid Tissue proteinlysate prepared from a tissue that had been formalin fixed and paraffinembedded. Data from such as assay indicates the presence of the uniqueSRM/MRM signature peak for this MGMT peptide in the formalin fixedsample.

Specific transition ion characteristics for this peptide are used toquantitatively measure a particular MGMT peptide in formalin fixedbiological samples. These data indicate absolute amounts of this MGMTpeptide as a function of molar amount of the peptide per microgram ofprotein lysate analyzed. Assessment of MGMT protein levels in tissuesbased on analysis of formalin fixed patient-derived tissue can providediagnostic, prognostic, and therapeutically-relevant information abouteach particular patient. In one embodiment, this disclosure describes amethod for measuring the level of the 6-O-methylguanine-DNAmethyltransferase protein (MGMT) in a biological sample, comprisingdetecting and/or quantifying the amount of one or more modified orunmodified MGMT fragment peptides in a protein digest prepared from thebiological sample using mass spectrometry; and calculating the level ofmodified or unmodified MGMT protein in the sample; and wherein the levelis a relative level or an absolute level. In a related embodiment,quantifying one or more MGMT fragment peptides comprises determining theamount of the each of the MGMT fragment peptides in a biological sampleby comparison to an added internal standard peptide of known amount,wherein each of the MGMT fragment peptides in the biological sample iscompared to an internal standard peptide having the same amino acidsequence. In some embodiments the internal standard is an isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinationsthereof.

The method for measuring the level of the MGMT protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) maybe used as a diagnostic and/or prognostic indicator of cancer in apatient or subject. In one embodiment, the results from measurements ofthe level of the MGMT protein may be employed to determine thediagnostic stage/grade/status of a cancer by correlating (e.g.,comparing) the level of MGMT protein found in a tissue with the level ofthat protein found in normal and/or cancerous or precancerous tissues.

Because both nucleic acids and protein can be analyzed from the sameLiquid Tissue™ biomolecular preparation it is possible to generateadditional information about disease diagnosis and drug treatmentdecisions from the nucleic acids in same sample upon which proteins wereanalyzed. For example, if the MGMT protein is expressed by certain cellsat increased levels, when assayed by SRM the data can provideinformation about the state of the cells and their potential foruncontrolled growth, potential drug resistance and the development ofcancers can be obtained. At the same time, information about the statusof the MGMT genes and/or the nucleic acids and proteins they encode(e.g., mRNA molecules and their expression levels or splice variations)can be obtained from nucleic acids present in the same Liquid Tissue™biomolecular preparation can be assessed simultaneously to the SRManalysis of the MGMT protein. Any gene and/or nucleic acid not from theMGMT and which is present in the same biomolecular preparation can beassessed simultaneously to the SRM analysis of the MGMT protein. In oneembodiment, information about the MGMT protein and/or one, two, three,four or more additional proteins may be assessed by examining thenucleic acids encoding those proteins. Those nucleic acids can beexamined, for example, by one or more, two or more, or three or more of:sequencing methods, polymerase chain reaction methods, restrictionfragment polymorphism analysis, identification of deletions, insertions,and/or determinations of the presence of mutations, including but notlimited to, single base pair polymorphisms, transitions, transversions,or combinations thereof.

The invention claimed is:
 1. A method for measuring the level of the6-O-methylguanine-DNA methyltransferase protein (MGMT) in a humanbiological sample of formalin-fixed tissue, comprising detecting andquantifying the amount of a MGMT fragment peptide in a protein digestprepared from said biological sample, using mass spectrometry, whereinsaid MGMT fragment peptide is the peptide having the sequence of SEQ IDNO:1; and calculating the level of modified or unmodified MGMT proteinin said sample; and wherein said level is a relative level or anabsolute level.
 2. The method of claim 1, further comprising the step offractionating said protein digest prior to detecting and quantifying theamount of said MGMT fragment peptide.
 3. The method of claim 2, whereinsaid fractionating step is selected from the group consisting of liquidchromatography, nano-reversed phase liquid chromatography, highperformance liquid chromatography, or reverse phase high performanceliquid chromatography.
 4. The method of claim 1, wherein said proteindigest comprises a protease digest.
 5. The method of claim 4, whereinsaid protein digest comprises a trypsin digest.
 6. The method of claim1, wherein the tissue is paraffin embedded tissue.
 7. The method ofclaim 1, wherein the tissue is obtained from a tumor.
 8. The method ofclaim 1, wherein quantifying the MGMT fragment peptide comprisescomparing an amount of said MGMT fragment peptide in one biologicalsample to the amount of the same MGMT fragment peptide in a differentand separate biological sample.
 9. The method of claim 1, whereinquantifying said MGMT fragment peptide comprises determining the amountof said MGMT fragment peptide in a biological sample by comparison to anadded internal standard peptide of known amount, wherein said MGMTfragment peptide in the biological sample is compared to an internalstandard peptide having the same amino acid sequence.
 10. The method ofclaim 9, wherein the internal standard peptide is an isotopicallylabeled peptide.
 11. The method of claim 10, wherein the isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H and combinationsthereof.
 12. The method of claim 1, further comprising selecting for thesubject from which said biological sample was obtained a treatment basedon the presence, absence, or amount of said MGMT fragment peptide or thelevel of MGMT protein.
 13. The method of claim 12, further comprisingadministering to the patient from which said biological sample wasobtained a therapeutically effective amount of a therapeutic agent,wherein the therapeutic agent and/or amount of the therapeutic agentadministered is based upon amount of said MGMT fragment peptide or thelevel of MGMT protein.
 14. The method of claim 13, wherein saidtherapeutic agent binds the MGMT protein and/or inhibits its biologicalactivity.